Hantaviruses (HVs) infect the endothelial cell (EC) lining of capillaries and nonlytically cause vascular leakage. Andes virus (ANDV) and Sin Nombre virus (SNV) infect virtually all pulmonary ECs resulting in acute pulmonary edema that is 35% fatal (HV pulmonary syndrome-HPS). However, ANDV is the only HV spread person to person and the only HV that causes lethal HPS-like disease in Syrian hamsters. We found that the ANDV nucleocapsid (N) protein uniquely regulates interferon (IFN) induction, suggesting that ANDV encodes a novel virulence determinant. HVs are inhibited by IFN added prior to or early after infection, and in order to replicate in human ECs pathogenic HVs regulate early IFN induction. Except for PHV, HV Gn proteins contain cytoplasmic tail (GnT) elements that inhibit RIG-I/TBK1 directed IRF3 phosphorylation and IFN? induction. In contrast, N proteins from SNV, NY-1V, HTNV and PHV fail to inhibit RIG-I/TBK1 directed IFN induction. Unexpectedly, the ANDV N protein inhibited RIGI/TBK1 directed IFN induction. Yet, IFN signaling was not regulated by N protein from MAPV, a S. American ANDV homologue not linked to human disease. Thus only ANDV expresses an N protein that regulates RIGI/MDA5/MAVS and TBK1 IFN signaling responses. Further analysis determined that ANDV N inhibits TBK1 autophosphorylation while Gn prevents activated pTBK1 from phosphorylating IRF3. Thus ANDV uniquely expresses an IFN regulating N protein that may act in concert with Gn to regulate sequential TBK1 signaling steps. Consistent with this, N and GnGc proteins localize to the ER/cisGolgi where TRAF3/TBK1/TANK complexes are regulated by a plethora of factors. Our recent proteomics analysis of N interactions with EC proteins defined TRIM21 as a putative ANDV N binding partner. TRIM21 regulates TBK1-IRF3 signaling responses suggesting that ANDV N may target TRIM21 to inhibit IFN induction. This further suggests that Gn proteins may independently or synergistically target TRIM21 functions to inhibit TBK1-IRF3 signaling. Our data defines N protein as a novel ANDV-specific virulence determinant. This may explain why ANDV uniquely causes viremia and lethal HPS-like disease in Syrian hamsters and is the only HV that is spread person to person. Our recent findings, the development of ANDV reverse genetics and our partnership with Jay Hooper (USAMRIID) permit us to propose defining N and Gn mutations that attenuate ANDV and prevent lethal HPS in Syrian hamsters. We propose defining IFN regulating virulence determinants within HV N and Gn proteins and mechanisms by which N/Gn regulate RIG-I/TBK1 signaling pathways. We will also determine whether mutating IFN regulating elements within N and Gn or reasserting ANDV with MAPV 1) reduces ANDV replication in human ECs; 2) attenuates ANDV directed HPS in the lethal Syrian hamster HPS model and 3) protects hamsters from lethal ANDV infection. These studies are likely to develop attenuated ANDVs as potential vaccines.